Age-related (senile) cataract is principally a crystallin protein aggregation and subsequent precipitation disease that occurs over the time frame of several years. During cataract development, the increased sizes of aggregated and cross-linked crystallin multimers become so large that they finally become water insoluble and cause lens opacity. Deamidation and truncations of crystallins are identified as the most abundant post-translational modifications (PTMs), and are believed to be the major causative factors in age-related cataract development. However, the mechanisms of cataract development by either deamidated- or truncated-crystallins are still unknown. Based on our extensive results, we have hypothesized that deamidated and truncation of crystallins synergistically cause cataract by producing aggregates that deposit on lens membranes and lead to fiber cells degeneration. The aggregation process is initiated by truncated crystallin fragments that form amyloid fibril-type of complexes alone and/or with fragments of deamidated crystallins, filensin and phakinin. In this process, ?A3-protease-derived fragments of deamidated crystallins play a major role. We plan to test the above hypothesis by seeking answers to the following three questions: (1) What is molecular mechanism of cataractogenesis in ?A-N101D transgenic mouse model? Our first ever available transgenic mouse model with ?A-N101D transgene develops cortical cataract at about 7-month of age. Because several preceding phenotypic changes trigger cataract development at 7-months of age in transgenic mice relative to wild type mice, the mouse model will be used to elucidate the molecular mechanism of deamidated ?AN101D-induced cataract development. (2) How is the active site of ?A3 protease regulated and activated in vivo to proteolyze crystallins? We will determine how the active site ?A3 protease is regulated intrinsically by its N terminal arm and extrinsically by ?A- and ?B-crystallins as inhibitors, and how ?A3-protease preferentially proteolyzes deamidated and/or truncated crystallins relative to unmodified crystallins in vitro and in ?A-N101D transgenic mice. (3) What is the molecular mechanism of aggregation of fragments of crystallins (derived via proteolysis by ?A3-protease) and phakinin and filensin with a deamidated 4-kDa ?B fragment? It will be determined whether amyloidogenesis is the mechanism of aggregation among crystallin fragments (derived via proteolysis by ?A3-protease), fragments of filensin and phakinin, and an in vivo existing deamidated 4-kDa ?B fragment. Because mainly human lenses will be used in these studies, the findings will be relevant to in vivo mechanism of age-related cataract development. The results will be of significant therapeutic value to delay the development and progression of age-related cataract.
The overall goal of the proposal is to elucidate molecular mechanism of development of human age-related cataract due to the two most prevalent but synergistic in vivo post-translational modifications of crystallins: deamidation and truncation. Th proposed studies will provide answers to the central question about the mechanism of how deamidated and truncated crystallins aggregate and cause lens opacity. The findings would be used for therapeutic purpose to delay human age-related cataract.
|Srivastava, O P; Srivastava, K; Chaves, J M et al. (2017) Post-translationally modified human lens crystallin fragments show aggregation in vitro. Biochem Biophys Rep 10:94-131|
|Chaves, Jose M; Gupta, Ratna; Srivastava, Kiran et al. (2017) Human alpha A-crystallin missing N-terminal domain poorly complexes with filensin and phakinin. Biochem Biophys Res Commun 494:402-408|
|Hegde, Shylaja; Kesterson, Robert A; Srivastava, Om P (2016) CRY?A3/A1-Crystallin Knockout Develops Nuclear Cataract and Causes Impaired Lysosomal Cargo Clearance and Calpain Activation. PLoS One 11:e0149027|
|Tiwary, Ekta; Hegde, Shylaja; Purushotham, Sangeetha et al. (2015) Interaction of ?A3-Crystallin with Deamidated Mutants of ?A- and ?B-Crystallins. PLoS One 10:e0144621|
|Hegde, Shylaja M; Srivastava, Kiran; Tiwary, Ekta et al. (2014) Molecular mechanism of formation of cortical opacity in CRYAAN101D transgenic mice. Invest Ophthalmol Vis Sci 55:6398-408|
|Gupta, Ratna; Asomugha, Chinwe O; Srivastava, Om P (2011) The common modification in alphaA-crystallin in the lens, N101D, is associated with increased opacity in a mouse model. J Biol Chem 286:11579-92|
|Asomugha, C O; Gupta, R; Srivastava, O P (2010) Identification of crystallin modifications in the human lens cortex and nucleus using laser capture microdissection and CyDye labeling. Mol Vis 16:476-94|
|Gupta, R; Chen, J; Srivastava, O P (2010) A serine-type protease activity of human lens ?A3-crystallin is responsible for its autodegradation. Mol Vis 16:2242-52|
|Gupta, Ratna; Srivastava, Om P (2009) Identification of interaction sites between human betaA3- and alphaA/alphaB-crystallins by mammalian two-hybrid and fluorescence resonance energy transfer acceptor photobleaching methods. J Biol Chem 284:18481-92|
|Srivastava, K; Gupta, R; Chaves, J M et al. (2009) Truncated human betaB1-crystallin shows altered structural properties and interaction with human betaA3-crystallin. Biochemistry 48:7179-89|